The last several decades have seen tremendous progress in the understanding of biological processes. Despite these advances, research in many important fields, such as immunology and cancer biology, has made it increasingly clear that bulk measurements can mask characteristics of individual cells or subsets of cells. Such individual cells and small subsets of cells may contribute significantly to biological processes, yet may not be identical to the population average measured by existing techniques. In addition, interactions between individual players may not be resolved if only an average behavior is studied. As a result, traditional methods may draw a misleading picture of dynamic responses of cells to the given perturbations of their biological environments, necessitating development of technologies for single-cell analysis. Moreover, inefficiencies in sample handling and data collection inherent in current flow-based profiling methods (e.g., flow cytometry) limit comprehensive phenotyping of the scarce cells recovered from tissue samples.
Conventional slide-based cytometry can efficiently provide capture of all cells in the sample in a first step, preventing cell loss during cell staining and data acquisition. However, current methods of acquiring images of the captured cells, such as laser scanning slide cytometry and multi-parameter confocal microscopy, have (1) lagged on the number of channels available on state-of-the art flow cytometers and (2) are costly, which restrict their availability primarily to core facilities. Moreover, while conventional methods using iterative staining have expanded the number of markers that can be detected, they are both labor and time intensive.
Further, typical slide-based cytometry methods require the cells to be fixed to the slide, thereby preventing further analysis of these precious cell samples using functional assays, which are critical for understanding the role of these cells in tissue-restricted immune responses.
There is a need for the development of efficient methodologies for interrogating cells (e.g., lymphocytes, leukocytes, tumor cells, stromal cells, neuronal cells, cell lines (e.g., CHO cells, NS0 cells), stem cells, embryos, and the like) present in scarce cell samples to advance understanding of clinical responses to the growing number of experimental interventions targeting tissue in the fields of cancer immunotherapy, autoreactive bowel disorders, allergy, infectious disease, multiple sclerosis, neuroimmunological disease, and HIV. Development of such methods must have the ability to image a large area for increased throughput, have a large spectral depth (e.g., 10-30 color channels for 10-30 markers), automatically scan large area for scarce cells, pick the scarce cells, and maintain cell viability for further functional characterization.